During the past decade, major efforts using genomic and genetic approaches have identified two major classes of innate immune receptors for sensing microbial nucleic acids: Toll-like receptors (TLR: TLR3, 7, 8, 9) and retinoic acid-inducible gene I-like helicases (RLH: RIG-I, LGP2, MDA-5). However, genomic and genetic approaches have left a major gap in our understanding on how these receptors bind nucleic acids and whether additional receptors or coreceptors exist. We decided to open this Black Box by taking a biochemical approach to directly isolate and characterize microbial nucleic acid-binding proteins in human plasmacytoid dendritic cells (pDCs) by CpG-DNA pull down and mass spectrometric peptide sequencing experiments. We identified two novel members of the DExD/H-box helicase family DHX36 and DHX9 that specifically bind CpG- A and CpG-B, respectively, in human pDCs. Knocking down DHX36 expression by siRNA in a pDC cell line was associated with over 50% reduction in type 1 IFN responses and abolished IRF7 nuclear translocation induced by CpG-A and knocking down DHX9 expression was associated with a diminished TNF and IL-6 response and blocking of NF-kB p50 nuclear translocation induced by to CpG-B. The DExD/H helicase family has over 50 members. Based on these preliminary data and the fact that the other viral sensors, including RIG- I-like helicases (RIG-I, LGP2, MDA-5) and Dicer, all belong to the DExD/H helicase family, this proposal will test our central hypotheses: 1) the DExD/H-box helicases DHX36 and DHX9 may represent novel TLR9- independent DNA sensors in pDCs; and 2) the DExD/H helicase family (59 members) may play a much broader role in anti-viral innate immunity than previously thought. )